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Thiol-catalyzed formation of NO-ferroheme regulates intravascular NO signaling

Nature Chemical Biology volume 19, pages 1256–1266 (2023)Cite this article

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Abstract

Nitric oxide (NO) is an endogenously produced signaling molecule that regulates blood flow and platelet activation. However, intracellular and intravascular diffusion of NO are limited by scavenging reactions with several hemoproteins, raising questions as to how free NO can signal in hemoprotein-rich environments. We explore the hypothesis that NO can be stabilized as a labile ferrous heme–nitrosyl complex (Fe2+-NO, NO-ferroheme). We observe a reaction between NO, labile ferric heme (Fe3+) and reduced thiols to yield NO-ferroheme and a thiyl radical. This thiol-catalyzed reductive nitrosylation occurs when heme is solubilized in lipophilic environments such as red blood cell membranes or bound to serum albumin. The resulting NO-ferroheme resists oxidative inactivation, is soluble in cell membranes and is transported intravascularly by albumin to promote potent vasodilation. We therefore provide an alternative route for NO delivery from erythrocytes and blood via transfer of NO-ferroheme and activation of apo-soluble guanylyl cyclase.

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Fig. 1: Basis spectra and reaction kinetics of GSH-assisted NO-ferroheme formation in anaerobic MeOH:PBS buffer.
Fig. 2: GSH accelerates the reaction of NO and ferric heme in suspended RBC membrane white ghosts under anaerobic conditions.
Fig. 3: NO-ferroheme formation via GSH-catalyzed reductive nitrosylation of ferric heme in serum albumin.
Fig. 4: Effects of NO-ferroheme albumin on platelet activation.
Fig. 5: Changes in murine MAP by NO-ferroheme in albumin from glutathione-catalyzed reductive nitrosylation.
Fig. 6: Changes in murine MAP by NO-ferroheme prepared via ferrous heme generation by sodium dithionite and NO addition in albumin.

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Data availability

All the data generated in this study are available within the main text and the Supplementary Information file. Data are also available from the corresponding author upon request. The source datasets generated and analyzed during the current study are available on Dryad at https://doi.org/10.5061/dryad.mw6m9062d. Source data are provided with this paper.

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Acknowledgements

We thank M. Guthold and H. Lee for help with DIC microscopy. This work was supported by NIH grants R01 HL125886 (J.T. and M.T.G.), R01 HL098032 (M.T.G. and D.B.K.-S.), K08 HL136857 (J.J.R.) and DOD grant W81XWH2210198 (J.T. and J.J.R.).

Author information

Author notes

  1. These authors contributed equally: Anthony W. DeMartino, Laxman Poudel.

  2. These authors jointly supervised this work: Mark T. Gladwin, Daniel B. Kim-Shapiro.

Authors and Affiliations

  1. Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, USA

    Anthony W. DeMartino, Qinzi Xu, Jason J. Rose & Mark T. Gladwin

  2. Department of Physics, Wake Forest University, Winston-Salem, NC, USA

    Laxman Poudel, Swati Basu, Elmira Alipour, Yiyang Jiang & Daniel B. Kim-Shapiro

  3. Heart, Lung, Blood, and Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA, USA

    Matthew R. Dent, Xiukai Chen, Brendan S. Gladwin & Jesús Tejero

  4. Division of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA, USA

    Jesús Tejero

  5. Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA

    Jesús Tejero

  6. Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA, USA

    Jesús Tejero

  7. Translational Science Center, Wake Forest University, Winston-Salem, NC, USA

    Swati Basu & Daniel B. Kim-Shapiro

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Contributions

A.W.D. designed and performed experiments, analyzed data and wrote the initial and final drafts of the paper. L.P. performed and helped design experiments, including the initial ones discovering the thiol-based catalysis, and analyzed data. M.R.D., X.C., Q.X., B.S.G., J.T., S.B., E.A., J.J.R. and Y.J. also performed experiments and/or analyzed data. All authors reviewed and edited the manuscript. M.T.G. and D.B.K.-S. directed the research and designed experiments, interpreted data and share senior authorship.

Corresponding authors

Correspondence to Mark T. Gladwin or Daniel B. Kim-Shapiro.

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Competing interests

A.W.D., J.J.R., M.T.G., J.T., M.R.D., D.B.K.-S. and L.P. have a provisional patent filed at the University of Pittsburgh (application no. 63/420,030), related to the creation and use of NO-ferroheme. Though not related directly to NO-ferroheme, A.W.D., J.T., J.J.R., M.T.G., M.R.D. and Q.X. are co-inventors on patents and patent applications directed at the use of heme proteins as therapeutic agents. M.T.G., D.B.K.-S. and J.J.R. are co-inventors on patent and/or patent applications related to sodium nitrite as a therapeutic. Some of these patents are licensed to Globin Solutions, Inc. J.J.R., M.T.G. and J.T. are shareholders of Globin Solutions. J.J.R. and J.T. are officers and directors of Globin Solutions. A.W.D. is a consultant of Globin Solutions. M.T.G. is a consultant, director and scientific advisor to Globin Solutions. The other authors declare no competing interests.

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Nature Chemical Biology thanks Arnab Ghosh, Emil Martin and Douglas Thomas for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Scheme depicting electron transfer to generate NO-ferroheme.

Ferric labile heme reacts with NO to generate a labile nitrosyl ferric heme, which readily is reduced by 1 electron by a small thiol like glutathione (GSH). The mechanism of transfer may follow an inner sphere mechanism (I.S.) where the GSH binds the nitrosyl ferric heme first then transfers the electron, or an outer sphere mechanism (O.S) where binding does not occur. In the presence of excess NO, the generated thiyl radical from either mechanism will react with NO to yield S-nitrosoglutathione (GSNO).

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–7.

Reporting Summary

Supplementary Data 1

Data for Supplementary Fig. 1. Example NO traces from NO chemiluminescence analyzer, source data.

Supplementary Data 2

Data for Supplementary Fig. 2. Formation of NO-ferroheme via GSH-catalyzed reductive nitrosylation of ferric heme in albumin at different NO concentrations, source data.

Supplementary Data 3

Data for Supplementary Fig. 3. Other transfers of NO-ferroheme, source data.

Supplementary Data 4

Data for Supplementary Fig. 4. NO-ferroheme albumin stability in the presence of an equivalent of oxyhemoglobin, source data.

Supplementary Data 5

Data for Supplementary Fig. 5. Other relevant platelet control experiments, source data.

Supplementary Data 6

Data for Supplementary Fig. 6. Elimination of nitrite as potential vasodilating species during administration of NO-ferroheme albumin solution, source data.

Supplementary Data 7

Data for Supplementary Fig. 7. Typified flow cytometry gating for platelet sorting and platelet activation determination.

Source data

Source Data Fig. 1

Data for Fig. 1.

Source Data Fig. 2

Data for Fig. 2.

Source Data Fig. 3

Data for Fig. 3.

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Data for Fig. 4.

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Data for Fig. 5.

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Data for Fig. 6.

Source Data Extended Data Fig. 1

Chemdraw file for Extended Data Fig. 1.

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DeMartino, A.W., Poudel, L., Dent, M.R. et al. Thiol-catalyzed formation of NO-ferroheme regulates intravascular NO signaling. Nat Chem Biol 19, 1256–1266 (2023). https://doi.org/10.1038/s41589-023-01413-3

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